The gas, carbon monoxide (CO), is produced physiologically by catabolism of heme via heme oxygenase. One of the isoforms of heme oxygenase, HO-2, is expressed in highest concentration in the brain where it resides in neurons, vascular endothelium and smooth muscle. It generates CO from cellular heme. This application is based on preliminary data that suggest carbon monoxide (CO) is an endogenous mediator in the newborn cerebral circulation. These preliminary data include detection of heme oxygenase 2 (HO-2) in the cerebral vasculature, cerebrovascular dilator responses to exogenously applied CO and to heme oxygenase substrate, and measurements of CO production by cerebral microvessels and endothelium. The likelihood that CO will prove to be a physiologically significant intercellular messenger molecule is high. The research proposed is designed to pursue the unifying hypothesis that CO is an integral component of microvascular control in neonatal cerebral circulation. To test this hypothesis, three specific aims will be addressed in newborn pigs, in vivo, and using cells isolated from newborn pigs, in primary culture: 1. Determine the functional significance of CO in regulation of cerebral microvascular tone, 2. Localize and characterize heme oxygenase of the neonatal cerebral vasculature, 3. Investigate mechanisms of CO induced modifications of cerebral microvascular tone. To accomplish these aims, techniques allowing investigation of intact cerebral microcirculation, isolated cerebral microvessels, and primary culture of cells from newborn pigs will be employed. Such research is unique by studying intact cerebral circulation and investigating, at the cellular and molecular levels, the mechanisms responsible for controlling the production of the mediator, CO, and the mechanisms by which CO can affect vascular tone. Cranial windows allow observation of cerebral circulation, collection of cortical periarachnoid fluid, and topical application of agonists, precursors and inhibitors. Levels and cellular distribution of heme oxygenase protein expression as well as enzyme activity and cellular mechanisms for controlling that activity will be examined. The cellular mechanisms by which CO may modify vascular tone will be studied at the level of membrane potential, ion channels, and second messengers. Disorders of the cerebral circulation in the newborn period are major causes of morbidity and mortality and can result in life long disabilities in survivors. Control of cerebrovascular circulation is easily impaired by pathological conditions. Therefore, better understanding of mechanisms of cerebromicrovascular humoral communication in newborns is needed badly. 3ERFORMANCE SITE(S) (organization, city, state) The University of Tennessee, Memphis Memphis, TN 38163 KEY PERSONNEL. See instructions on Page 11. Use continuationpages as neededto provide the required information in the format shown below. Name Organization Role on Project Charles W. Leffler, Ph.D. The University of Tennessee Principal Investigator Helena Parfenova, Ph.D. The University of Tennessee Co-Principal Investigator David Mendelowitz, Ph.D. The University of Tennessee Co-Investigator PHS 398 (Rev. 4/98) Page 2 BB Number pages consecutively at the bottom throughoutthe application. Do not use suffixes such as 3a, 3b. cc Princ fivestigator/Program Director (Last, first, middle): 5SW. Type the name of the principal investigator/program director at the top of each printed page and each continuation page. (Fortype specifications, see instructions on page6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,